Compositions and methods for affecting virulence determinants in bacteria

ABSTRACT

A novel sarR gene and sarR gene product which down regulates the expression of sarA and the resulting virulence determinants in  Staphylococcus aureus  is provided. Methods for modulating the expression of sarA and virulence determinants are also provided. A preferred embodiment of the present invention provides structural information relating to the gene product and enables the identification and formulation of lead compounds and reducements for treating and preventing infections by  S. aureus  and related bacteria.

This application claims priority to U.S. Provisional Application Ser.No. 60/261,233, filed Jan. 12, 2001, U.S. Provisional Application Ser.No. 60/261,607, filed Jan. 12, 2001, and U.S. Provisional ApplicationSer. No. 60/289,601, filed May 8, 2001. These applications are herebyincorporated by references herein in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of molecularbiology. More particularly, certain embodiments concern methods andcompositions comprising DNA segments and protein derived fromStaphyloccocus aureus and other bacterial species. The present inventionalso relates to the three-dimensional structure of proteins derived fromS. aureus and other bacterial species and methods of identifying anddeveloping pharmaceuticals using, among other things, drug screeningassays.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byArabic numerals within parentheses. Full citations for thesepublications is found at the end of the specification immediatelypreceding the claims. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application.

Bacterial infections are a serious problem in humans. In the pastdecade, the number of “supergerms” that resist treatment has increaseddramatically. Unfortunately, the very same arsenal of drugs used toovercome these microbes helped give rise to antibiotic-resistant strainsof bacteria. Of great importance are several antibiotic-resistant andsometimes fatal bacteria including S. aureus, Pseudomonas aeruginosa(pneumonia), and Enterococcus faecalis (urinary tract and bloodinfections).

For most healthy people, these antibiotic-resistant bacteria are notlife-threatening. The immune system, the body's natural defense againstmicrobes, usually fights off disease-causing bacteria. However, whenbacteria attack people with weakened immune systems they can be deadly.Hailed as miracle drugs, antibiotics have cured thousands of bacterialinfections, from acne to strep throat to ear infections. Today, thereare more than 100 types of antibiotics on the U.S. market. Due toincreasing resistance to antibiotics, however, new treatments are stillneeded.

In particular, S. aureus infections have been problematic to treat. S.aureus are non-mobile, non-sporulating gram-positive cocci 0.5-1.5 μm indiameter, that occur singly and in pairs, short chains, and irregularthree-dimensional grape-like clusters. S. aureus can grow over a widerange of environmental conditions, but they grow best at temperaturesbetween 30° C. and 37° C. and at a neutral pH. They are resistant todesiccation and to chemical disinfection, and they tolerate NaClconcentrations up to 12%. It has been found that the growth of S. aureusbecomes unusually sensitive to high NaCl concentrations (by decreasingCa²⁺ concentration) in growth media allowing for autolysis (29).

S. aureus can cause a wide spectrum of infections ranging fromsuperficial abscesses; pneumonia and endocarditis to sepsis (4). Theability of S. aureus to cause a multitude of human infections is due, inpart, to an impressive array of extracellular and cell-wall associatedvirulence determinants that are coordinately expressed in this organism(51). The coordinate expression of many of these virulence determinantsin S. aureus and other bacteria is regulated by global regulatoryelements such as sarA and agr (15, 34). These regulatory elements, inturn, control the transcription of a wide variety of unlinked genes manyof which have been implicated in pathogenesis.

The global regulatory locus agr encodes a two-component, quorum sensingsystem that is involved in the generation of two divergent transcripts,RNAII and RNAIII, from two distinct promoters, P2 and P3, respectively.RNAIII is the regulatory molecule of the agr response, hence responsiblefor the up-regulation of extracellular protein production anddown-regulation of cell-wall associated protein synthesis during thepostexponential phase (39,49). The RNAII molecule, driven by the P2promoter, encodes a four-gene operon, agrBDCA, with AgrC and AgrAcorresponding to the sensor and activator proteins of a two componentregulatory system. Additionally, agrD, in concert with agrB,participates in the generation of an octapeptide with quorum sensingfunctions (31,41). The autoinducing peptide would stimulate thetranscription of the agr regulatory molecule RNAIII which ultimatelyinteracts with target genes to modulate transcription (49) and possiblytranslation (44).

In contrast to agr, the sarA locus activates the synthesis of bothextracellular (e.g. α- and β-hemolysins) and cell-wall proteins (e.g.fibronectin binding protein) in S. aureus (15). The sarA locus iscomposed of three overlapping transcripts [sarA P1 (0.56 kb), sarA P3(0.8 kb) and sarA P2 (1.2 kb) transcripts], each with a common 3′ endbut initiated from three distinct promoters (P1, P3 and P2 promoters).Due to their overlapping nature, each of these transcripts encodes themajor 372-bp sarA gene, yielding the 14.5 kDa sarA protein (2). DNAfootprinting studies have shown that the sarA protein binds to thepromoters of several target genes (19) including agr, hla (alphahemolysin gene), spa (protein A gene) and fnbA (fibronectin bindingprotein A gene), thus implicating sarA as a regulatory molecule that canmodulate target gene transcription via both agr-dependent andagr-independent pathways (9,19,20). With agr-dependent pathway of targetgene activation, the sarA protein binds to the agr promoter to stimulateRNAIII transcription and RNAIII, in turn, interacts with target genes(e.g. hla) to modulate transcription. With sarA-dependent butagr-independent pathway, the SarA protein will interact directly withtarget-gene promoters (e.g. hla and spa) (19) to control genetranscription. Deletion and promoter fusion analyses indicates that theregions upstream of the sarA P2 and between the P1 and P3 promoters havea modulating role in sarA expression, possibly by controllingtranscription from the sarA P1 promoter, the predominant promoter withinthe sarA locus (10,39) (FIG. 1A).

A great need exists for methods and compositions which can affect orregulate the virulence of bacteria, such as the expression of sarA andthe resultant virulence determinants of S. aureus and other bacteria.

SUMMARY OF THE INVENTION

The present invention provides a new genetic locus of S. aureus andother bacteria. The gene at this locus is referred to herein as sarR.The sarR gene is involved in the regulation and expression of virulencedeterminants in S. aureus and other bacteria.

The present invention provides a polynucleotide sequence of the sarRgene (SEQ. ID. NO: 1) which is involved in the regulation and expressionof virulence determinants in S. aureus and related bacteria. Alsoprovided is a sarR gene product, an isolated polypeptide which isinvolved in regulation and expression of virulence determinants in S.aureus and related bacteria.

An object of the present invention is to provide nucleic acid sequencesthat regulate the expression of virulence determinants in S. aureus andrelated bacteria. In a preferred embodiment the nucleic acid sequence isisolated from S. aureus and in another preferred embodiment the nucleicacid sequence is a sarR gene (SEQ. ID. NO: 1).

Another object of the present invention is to provide a polypeptidewhich regulates the expression of virulence determinants in S. aureusand other bacteria. In a preferred embodiment the polypeptide isisolated from S. aureus and in another preferred embodiment thepolypeptide is encoded by a sarR gene and correspond to the sequence setforth in SEQ ID NO: 2.

Vectors and host cells comprising nucleic acid sequences encoding thesepolypeptides and chemical entities that mimic or enhance the activity ofsuch polypeptides are further objects of the invention. These agents canbe used alone or in combination with antibacterial agents such aspenicillin to enhance the properties of such agents.

Another object of the present invention is to provide methods formodulating regulation and expression of virulence determinants in S.aureus and related bacteria to inhibit their growth and infectivity bycontacting the bacteria with an agent which interacts with sarA promoterregions.

Another object of the present invention is to provide a method ofinhibiting growth and infectivity of bacteria comprising contacting thebacteria with an agent which enhances the expression of the sarR gene,or the activity of a polypeptide encoded by the sarR. gene.

Another object of the present invention is to provide a kit foridentifying the presence of a sarR gene or a polypeptide encoded by asarR gene.

A further object of the present invention is to provide nucleic acidsequences from a mutant sarR gene in S. aureus and related bacteria, andpolypeptides encoded by a mutant sarR gene.

A further object of the invention is to provide a three dimensionalcrystal structure of the SarR protein.

A further object of the invention is to provide methods of screening forlead compounds which inhibit the expression of virulence determinants inS. aureus and related bacteria.

Another object of the present invention is to provide pharmaceuticalcompositions for use as anti-bacterial agents against S. aureus andother bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a schematic of the sar promoters and transcripts.

FIG. 1B shows the promoter region of sarR. The transcription start sitehas been mapped by primer extension to position 119. Examples of −10 and−35 promoter boxes are in bold and underlined.

FIG. 1C shows the alignment of SarR with SarA. Colon represents identitywhile a period indicates conservative substitution.

FIG. 2 demonstrates purification of sarR from the pET11b expressionvector.

FIG. 3A demonstrates PCR amplification of sarR-like genes in S. aureusstrains RN6390, Cowan I, DB and Newman, S. epidermidis, S. haemolyticusand S. saprophyticus.

FIG. 3B shows a Southern blot of the strains in FIG. 3A above strainrestricted with ClaI and probed with a 345-bp sarR probe (nt 208-552).

FIG. 3C shows a Northern blot of the total cellular RNA (10 μg each) ofthe strains of FIG. 3D probed with a sarR probe.

FIG. 3D shows cell lysates of the above strains immunoblotted ontonitrocellulose and probed with anti-sarR monoclonal antibody 2A7 at a1:2000 dilution.

FIG. 4 shows gel shift assays of end-labeled 32P fragment of sarA P1 (nt531-859) (2), P2 (nt 1-196) and P3 (nt 364-525) promoters.

FIG. 5 shows DNaseI footprinting assays of sarR with end-labeled 32P sarP2 (49-bp fragment), P1 (nt 531-859) and P1′ (nt 620-859) promoterfragments.

FIG. 6 shows the expression of sarR in parental strain RN6390 and itssar (ALC488) and agr (RN6911) mutants. FIG. 6A illustrates Northernblots of the sarR transcript in RN6390 and its isogenic sar and agrmutants. FIG. 6B illustrates ethidium bromide stain of an RNA gel priorto transfer to hybridization membrane. FIG. 6C illustrates theexpression of sarR on an immunoblot probed with anti-sarR antibody 2C7.

FIGS. 7A & B illustrate promoter activation of sarA P1 and combinedP2-P3-P1 promoters fused to a gfp_(uvr) reporter gene as evaluated in afluorescence spectrophotometer (FL600, BioTek Instrument).

FIG. 8 demonstrates the effect of the sarR mutation on sarA and agrexpression. FIG. 8A illustrates sarA expression during mid-log andlate-log phases and early stationary phases. FIG. 8B illustrates agrA(RNAII) transcription. It is a Northern blot of the RNAII (agrA probe)transcript in RN6390 and the sarR mutant (10 μg of total RNA each). TheagrA probe corresponds to nt 3830-4342 according to published sequence(23).

FIG. 9A illustrates a ribbon (8) diagram of the three-dimensionalstructure of the SarR-MBP fusion protein. The SarR dimer is at the topcolored Green and Yellow for each monomer, respectively, two MBPmolecules are at bottom colored Blue and Pink, respectively.

FIG. 9B illustrates a ribbon diagram of the three-dimensional structureof the SarR monomer. Starting from the N-terminus, α1 (6-24)→α2(32-44)→α1 (47-50→α3 (residue 51-58)→α4 (63-74)→β2 (79-83)'β3 (90-96)→α5(97-113). α2, ⊕1, α3, α4, β2, and β3 are defined as a “winged helixmotif”. α3→α4 represent a helix-turn-helix-motif.

FIG. 10A illustrates one view of the structure of the SarR dimer. OneSarR monomer is colored Green while the other is Yellow. This top viewof the concave side along the dimer 2-fold axis of the SarR dimer,subdomain1 contains β1, α3, α4, β2, β3 (labeled blue) from one monomer,subdomain 2 contains β1, α3, α4, β2, β3 (also labeled blue) from theother, subdomain 3 contains α1, α2, α5 (labeled red) from both monomers.

FIG. 10B illustrates a middle view perpendicular to the dimer 2-fold.

FIG. 10C illustrates a bottom view of the convex side of the SarR dimer.N1 and C1 are the N terminus and C terminus of molecule 1. N2 and C2 aretermini of molecule two.

FIG. 11 illustrates a sequence alignment of Sar homologs from S. aureus.The sequences are presented in one-letter amino acid code. Numbers atthe beginning of each line indicate amino acid positions relative to thestart of each protein sequence. Helices are indicated by rectangles,β-sheets are indicated by arrows, and loops are indicated by a line. “H”marked with Green represents residues that take part in dimerization.“A” marked with Red represents residues that may compose of theactivation motifs. “D” marked with Blue represents residues that isinvolved in the interactions of sarR with DNA. The sequence of DNAbinding motif from CAP is also aligned to the Sar proteins. Dot meansresidue or residues omitted, dash means residue or residues missed inCAP. SarS2 starts at 1 (actual position on SarS is 126).

FIGS. 12A and 12B illustrate the electrostatic potential surface of thesarR dimer calculated by GRASP (46), with charge +1 for Lys and Arg,charge −1 for Glu and Asp, and charge zero for all other residues. Thecolor bar from red to blue represents potential from negative topositive defined as in GRASP. The Blue represents positive chargedpotential, Red represents negative charged potential. FIG. 12Aillustrates the potential surface of the concave side of SarR dimer(similar orientation as FIG. 10A). The direct line distance of AB is˜65A; the distance of the inner surface covered by AB is 90° A. FIG. 12Billustrates the potential surface of the convex side of the SarR dimer(similar orientation as FIG. 10C). Two aspartic acid residues are fromone molecule, three glutamic acid residues from the other.

FIG. 13A illustrates the superposition of two “winged helix motifs”(subdomain 1 plus α2 from one monomer and subdomain 2 plus α2 fromanother monomer) of SarR dimer with the DNA binding domains ofcatabolite activating protein (CAP, PDB ID 1cgp); subdomains from SarRare marked Green and Yellow respectively; subdomains from CAP are markedBlue and Red; α4 and W1 interact with DNA at major grooves and minorgrooves respectively.

FIG. 13B illustrates the DNA binding model of SarR and DNA (similarorientation as FIG. 10A and FIG. 13A). The SarR dimer is superimposed tothat of CAP. The DNA structure is from the CAP-DNA complex structure(PDB ID 1cgp). The helix-turn-helix-motif binds to the major groovewhile the wing region (W1) conformation is slightly adjusted to fit inthe minor groove;

FIG. 13C illustrates a 90° orientation from view of FIG. 13B.

DETAILED DESCRIPTION OF THE INVENTION

If appearing herein, the following terms shall have the definitions setout below.

As used herein, the term “gene” refers to an assembly of nucleotidesthat encode a polypeptide, and includes cDNA and genomic DNA nucleicacids.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment is attached so as to bring about the replication ofthe attached segment.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase.

As used herein, the term “homologous” in all its grammatical formsrefers to the relationship between proteins that possess a “commonevolutionary origin,” including proteins from superfamilies andhomologous proteins from different species. Such proteins have sequencehomology as reflected by their high degree of sequence similarity.Preferably, homologous gene sequences will have at least 50% sequenceidentity (as defined by the maximal base match in a computer-generatedalignment of two nucleic acid sequences), more preferably at least 60%,and most preferably at least 80%. For polypeptide gene products of suchhomologous genes, generally the gene products also exhibit a significantdegree of amino acid sequence identity. Thus, for such polypeptideproducts of homologous genes, the amino acid sequences have at least 25%sequence identity over a sequence of 100 or more amino acids, morepreferably at least 40%, still more preferably at least 60%, and mostpreferably at least 80%. In addition, in the present context, theproducts of the homologous gene sequences are also involved inregulation of sara expression. Thus, the present invention applies aswell to bacteria having significant sequence, structural, or functionalhomology to the sarR gene or SarR protein.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin However, in common usage and in the instantapplication, the term “homologous,” when modified with an adverb such as“highly,” may refer to sequence similarity and not a common evolutionaryorigin.

The term “corresponding to” is used herein to refer similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

The term “a sequence essentially as set forth in SEQ ID NO: __” meansthat the sequence substantially corresponds to a portion of SEQ IDNO:__” and has relatively few amino acids that are not identical to, ora biologically functional equivalent of, the amino acids of SEQ IDNO:__. Accordingly, sequences that have between about 70% and about 80%;or more preferably, between about 81% and about 90%; or even morepreferably, between about 91% and about 99%; of amino acids that areidentical or functionally equivalent to the amino acids of SEQ ID NO:__will be sequences that are “essentially as set forth in SEQ ID NO:__.”

Typically, though not necessarily, a ligand in the context of thisinvention is a molecule with a molecular weight of less than 10,000daltons, more typically less than 5,000 daltons.

As used herein “related bacterial species” refer to bacterial specieshaving a gene corresponding to the sequence as essentially set forth inSEQ ID NO: 1, and analogs or homologs thereof. Related bacteriaaccording to the present invention include S. aureus strains RN6390,Newman, Cowan I and DB and S. saprophyticus.

The present invention provides a novel gene found in S. aureus and otherbacterial species. This gene is designated herein as sarR and isrepresented SEQ ID NO: 1. The present invention also provides for anovel gene corresponding to the sequence as essentially set forth in SEQID NO: 1, and analogs or homologs thereof.

The present invention also provides the sarR gene product designatedherein as the SarR protein represented in SEQ ID. NO. 2, and analogs orhomologs thereof. The SarR protein has a molecular weight ofapproximately 13.7 kDa and has a deduced basic pI of approximately 9.2.The sarR gene product is also characterized by a predominance of chargedresidues (34%).

The present invention also relates to newly discovered binding sites inthe sarA promoter system. These binding sites enable the SarR protein torepress sarA expression by preventing sarA from initiating theexpression of virulence factors

The present invention further provides a model for a SarA/SarRheterodimer which interferes with the function of the SarA homodimer.The present invention also provides alternative heterodimers whichinclude SarA/SarR peptidomimetic combinations and other combinationsresulting from lead compounds developed from structural analysis ofSarR.

The present invention also provides a sarR gene product orpeptidomimetic capable of directly binding to promoters of a virulencegene such as the hla promoter of the alpha hemolysin gene to providedirect inhibition of such virulence factors.

The present invention also provides a method of down; regulating sarA P1transcription to repress the expression of sarA. This function of thesarR gene product was demonstrated by the results of mutant studiesdisclosed herein. The sarR mutant expressed a much higher level of theSarA protein than the parent strain. Accordingly, the present inventionalso provides sarR mutant strains. These strains are useful asexperimental and diagnostic tools to specifically characterize the sarAexpression systems.

The present invention also provides a detailed three-diminsional (3-D)crystal structure of the SarR protein. The structural data demonstratethat the protein is a member of a new family of winged helix proteins.

The present invention provides methods for treating microbialpathogenesis by the administration of SarR analogs in the form of smallmolecule compounds which alter the functioning of a microbial sarAexpression. Reducing or eliminating the expression of sarA by suchanalogs can greatly alter the course and effects of a bacterialinfection. This treatment approach is distinct from most priorbactericidal or bacteristatic antimicrobial treatments which attemptedto either kill the microbial cells, or directly prevent them fromreproducing. The antimicrobial action of the compounds of these standardantimicrobial treatments is exerted both in vivo, in an infection, andin vitro, in a culture, unless some specific compensating factor(s) isprovided which allows survival or growth in the presence of theantimicrobial agent. In contrast, this invention is directed at theregulation of sarA expression which is involved in the pathogenesisprocess, but is not necessarily essential for microbial survival orgrowth in vitro. By selectively targeting the expression of virulencefactors and not directly killing the bacterium there is less pressure todevelop antimicrobial resistance. Because this type of therapy is lesslikely to alter the local flora, the incidence of superinfection (e.g.fungal or other bacterial infections such as pseudomonas orenterococcus) is reduced.

According to one aspect of the present invention, an analog library isprovided to produce a very large number of potential molecules forregulating the sarA expression system, and in general the greater:thenumber of analogs in the library, the greater the likelihood that atleast one member of the library will effectively regulate the sarAexpression system. Designed libraries following a particular templatestructure and limiting amino acid variation at particular positions aremuch preferred, since a single library can encompass all the designedanalogs and the included sequences will be known and presented inroughly equal numbers. By contrast, random substitution at only sixpositions in an amino acid sequence provides over 60 million analogs,which is a library size that begins to present practical limitationseven when utilizing screening techniques as powerful as phage display.Libraries larger than this would pose problems in handling, e.g.,fermentation vessels would need to be of extraordinary size, and moreimportantly, the likelihood of having all of the planned polypeptidesequence variations represented in the prepared library would decreasesharply. It is therefore preferred to create a designed or biasedlibrary, in which the amino acid positions designated for variation areconsidered so as to maximize the effect of substitution on the sarAregulation characteristics of the analog, and the amino acid residuesallowed or planned for use in substitutions are limited.

The use of replicable genetic packages, such as the bacteriophages, isone method of generating novel polypeptide entities that regulate sarAexpression. This method generally consists of introducing a novel,exogenous DNA segments into the genome of a bacteriophage (or otheramplifiable genetic package) so that the polypeptide encoded by thenon-native DNA appears on the surface of the phage. When the insertedDNA contains sequence diversity, then each recipient phage displays onevariant of the template (parental) amino acid sequence encoded by theDNA, and the phage population (library) displays a vast number ofdifferent but related amino acid sequences.

Such techniques make it possible not only to screen a large number ofpotential binding molecules but make it practical to repeat thebinding/elution cycles and to build secondary, biased libraries forscreening analog-displaying packages that meet initial criteria.

It is well-known to those normally skilled in the art that it ispossible to replace peptides with peptidomimetics. Peptidomimetics aregenerally preferable as therapeutic agents to peptides owing to theirenhanced bioavailability and relative lack of attack from proteolyticenzymes. Techniques of molecular modeling may be used to design apeptidomimetics which mimic the structure of the SarR peptide disclosedherein. Accordingly, the present invention also provides peptidomimeticsand other lead compounds which can be identified based on the dataobtained from structural analysis of the SarR protein disclosed herein.A potential SarR analog is examined through the use of computer modelingusing a docking program such as GRAM, DOCK, or AUTODOCK. This procedurecan include computer fitting of potential SarR analogs. Computerprograms can also be employed to estimate the attraction, repulsion, andsteric hindrance of an analog to a potential binding site. Generally thetighter the fit (e.g., the lower the steric hindrance, and/or thegreater the attractive force) the more potent the potential drug will besince these properties are consistent with a tighter binding constant.Furthermore, the more specificity in the design of a potential drug themore likely that the drug will not interfere with other properties ofthe sarA expression system. This will minimize potential side-effectsdue to unwanted interactions with other proteins.

Initially a potential SarR analog could be obtained by screening arandom peptide library produced by a recombinant bacteriophage, forexample, or a chemical library. A analog ligand selected in this mannercould be then be systematically modified by computer modeling programsuntil one or more promising potential ligands are identified.

Such computer modeling allows the selection of a finite number ofrational chemical modifications, as opposed to the countless number ofessentially random chemical modifications that could be made, and ofwhich any one might lead to a useful drug. Thus through the use of thethree-dimensional structure disclosed herein and computer modeling, alarge number of compounds is rapidly screened and a few likelycandidates can be determined without the laborious synthesis of untoldnumbers of compounds.

Once a potential SarR analog is identified it can be either selectedfrom a library of chemicals commercially available from most largechemical companies including Merck, GlaxoWelcome, Bristol Meyers Squib,Monsanto/Searle, Eli Lilly, Novartis and Pharmacia UpJohn, oralternatively the potential ligand is synthesized de novo. As mentionedabove, the de novo synthesis of one or even a relatively small group ofspecific compounds is reasonable in the art of drug design.

In a further aspect, this invention provides methods of treating abacterial infection by administering a pharmaceutical compositioncomprising a SarR analog, peptide, or peptidomimitic to inhibit theexpression of sarA. The present invention also provides a method forprophylactic treatment of a mammal, in particular a human, in order toprevent a bacterial infection. Such treatment comprises administering apharmaceutical preparation comprising a SarR analog, peptide, orpeptidomimetic to the mammal. Preferably such treatment would be usedwhen the patient is at risk of contracting or developing a bacterialinfection. Such a prophylactic treatment method may have particularbenefit, for example, for treating patients prior to surgicaloperations.

Pharmaceutical compositions according to the present invention maycomprise peptides and peptidomimetics of the present invention inassociation with a pharmaceutically acceptable carrier or excipient,adapted for use in human or veterinary medicine. The compositions maycontain from 0.001-99% of the active material. Such compositions may bepresented for use in conventional manner in admixture with one or morephysiologically acceptable carriers of excipients. The pharmaceuticalcompositions according to the invention may also contain other activeingredients such as antimicrobial agents, or preservatives. Thecompositions may optionally further contain one or more othertherapeutic agents which may, if desired, be a chemotherapeuticantiviral agent.

Pharmaceutically acceptable salts of the peptides of this invention maybe formed conventionally by reaction with an appropriate acid. Theadditional salts so formed from addition by acid may be identified byhydrochloric, hydrobromic, phosphoric, acetic, fumaric, salicylic,citric, lactic, mandelic, tartaric, oxalic, methanesulfonic, and thelike.

Thus, the peptides and peptidomimetics according to the presentinvention may be formulated for oral, buccal, parenteral, topical orrectal administration. In particular, these peptides and peptidomimeticsmay be formulated for injection or for infusion and may be presented inunit dose form in ampoules or in multidose containers with an addedpreservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g. sterile, pyrogen-free water,before use.

The present invention further provides a process for preparing apharmaceutical composition which comprises bringing a peptide orpeptidomimetic of the invention into association with a pharmaceuticallyacceptable excipient or carrier.

The dosage of the peptide or peptidomimetic used in the treatment willvary, depending on the seriousness of the disorder, the weight of thepatient, the relative efficacy of the peptide and the judgment of thetreating physician. Unit dosages may be administered more than once aday, e g., two or three times a day. Such therapy may extend for severalweeks, in an intermittent or uninterrupted manner, until the patient'ssymptoms are eliminated.

The present invention also provides pharmaceutical compositions whichcomprise a pharmaceutically effective amount of the peptides of thisinvention, or pharmaceutically acceptable salts thereof, and,preferably, a pharmaceutically acceptable carrier or adjuvant.Therapeutic methods of this invention comprise the step of treatingpatients in a pharmaceutically acceptable manner with those peptides orcompositions. Such compositions may be in the form of tablets, capsules,caplets, powders, granules, lozenges, suppositories, reconstitutablepowders, or liquid preparations, such as oral or sterile parenteralsolutions or suspensions.

In order to obtain consistency of administration, it is preferred that acomposition of the invention is in the form of a unit dose. The unitdose presentation forms for oral administration may be tablets andcapsules and may contain conventional expedients. For example bindingagents, such as acacia, gelatin, sorbitol, or polyvinylpyrrolidone;fillers, such as lactose, sugar, maize-starch, calcium phosphate,sorbitol or glycine; tabletting lubricants such as magnesium stearate;disintegrants, such as starch, polyvinylpyrrolidone, sodium starchglycolate or microcrystalline cellulose; or pharmaceutically acceptablewetting agents such as sodium lauryl sulfate.

The solid oral compositions may be prepared by conventional methods ofblending, filling, tabletting, or the like. Repeated blending operationsmay be used to distribute the active agent throughout those compositionsemploying large quantities of fillers. Such operations are, of course,conventional in the art. The tablets may be coated according to methodswell-known in normal pharmaceutical practice, in particular with anenteric coating.

Oral liquid preparations may be in the form of emulsions, syrups, orelixirs, or may be presented as a dry product-for reconstitution withwater or other suitable vehicle before use. Such liquid preparations mayor may not contain conventional additives. For example suspendingagents, such as sorbitol, syrup, methyl cellulose, gelatin,hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, orhydrogenated edible fats; emulsifying agents, such as sorbitanmonooleate or acacia; non-aqueous vehicles (which may include edibleoils), such as almond oil, fractionated coconut oil, oily estersselected from the group consisting of glycerine, propylene glycol,ethylene glycol, and ethyl alcohol; preservatives, for instance methylpara-hydroxybenzoate, ethyl para-hydroxybenzoate, n-propylparahydroxybenzoate, or n-butyl parahydroxybenzoate or sorbic acid; and,if desired, conventional flavoring or coloring agents.

For parenteral administration, fluid unit dosage forms may be preparedby utilizing the peptide and a sterile vehicle, and, depending on theconcentration employed, may be either suspended or dissolved in thevehicle. In preparing solutions, the peptides of this invention may bedissolved in water, whereas opiates used heretofore showed only marginalsolubility in aqueous media or physiological fluids. Once in solution,the peptide may be injected and filter sterilized before filling asuitable vial or ampoule and subsequently sealing the carrier or storagepackage. Adjuvants, such as a local anaesthetic, a preservative or abuffering agent, may be dissolved in the vehicle prior to use. Stabilityof the pharmaceutical composition may be enhanced by freezing thecomposition after filling the vial and removing the water under vacuum,e.g., freeze drying the composition. Parenteral suspensions may beprepared in substantially the same manner, except that the peptideshould be suspended in the vehicle rather than being dissolved. Asurfactant or wetting solution may be advantageously included in thecomposition to facilitate uniform distribution of the peptide.

Experimental Materials, Methods and Results

Bacterial Strains and Growth Conditions. As way of example, certainbacterial strains and plasmids used herein are listed in Table 1 asshown below. TABLE 1 Strains or plasmid References Comments S. aureusRN4220 (32) A mutant of 8325-4 that accepts DNA RN6390 (32) Laboratorystrain that maintains its hemolytic pattern when propagated on sheeperythrocyte agar (parental strain) RN6911 (34) An agr mutant of RN6390with an agr::tetM mutation ALC488  (9) A sarA mutant with a sarA::ermCmutation ALC1713 this study A sarR mutant of RN6390 with a sarR::ermCmutation Cowan I (17) A laboratory strain DB (10) A clinical bloodisolate previously used in adhesion and endocarditis studies Newman (29)A laboratory strain S. epidermidis A strain from the collection at theUtrecht University Hospital S. haemolyticus A strain from the collectionat the Utrecht University Hospital S. A strain from the collection atthe saprophyticus Utrecht University Hospital E. coli XL-1 blue (26) Ahost strain for cloning DH5α (26) A host strain for cloning PlasmidspCR2.1 Invitrogen E. coli cloning vector for direct cloning of PCRproducts pBluescript Stratagene E. coli cloning vector pUC18 (26) E.coli cloning vector pACYC177 New E. coli cloning vector England BioLabspCL52.1 (24) A temperature sensitive E. coli/S. aureus shuttle vectorpET11b Novagen Expression vector for E. coli pALC926 this study pUC18containing a 49-bp fragment upstream of the P2 promoter of the sarAlocus pALC1357 this study pET11b containing the 345-bp sarR gene at theNdcI/BamHI site pALC1361 this study pACYC177 with ˜4 kb ClaI fragmentcontaining the sarR region of RN6390 pALC1627 this study pBluescriptwith a 2.5 kb EcoRI/ClaI fragment containing the sarR gene subclonedfrom pALC1361 pALC1687 this study pBluescript with a 290-bp deletion ofthe sarR gene in pALC1627 pALC1696 this study pCL52.1 with a 290-bp sarRdeletion replaced by the ermC gene at the EcoRV/SalI site

Phage Φ11 was used as the transducing phage for S. aureus strains. S.aureus strain RN4220, a restriction-deficient derivative of strain8325-4 (47), was used as the initial recipient for the transformation ofplasmid construct by electroporation, following the protocol of Schenkand Laddaga (58).

S. aureus cells were grown at 37° C. with aeration in CYGP or 03GL broth(47,48) or tryptic soy broth supplemented with antibiotics whennecessary. 03GL and NYE agar (58) containing antibiotics were routinelyused for the selection of S. aureus transformants. Luria-Bertani mediumwas used for growing Escherichia coli. Antibiotics were used at thefollowing concentrations: erythromycin, 5 μg/ml; tetracycline, 5 μg/mland chloramphenicol, 10 μg/ml for S. aureus; and ampicillin, 50 μg/ml;chloramphenicol, 30 μg/ml; erythromycin, 200 μg/ml and spectinomycin, 75μg/ml for E. coli.

Cloning of the sarR gene and construction of the sarR mutant. The SarRprotein was partially purified from crude cell lysate by passing thelysate over a DNA-specific column containing a 49-bp DNA fragment (nt71-119) covalently linked to Sepharose (18). The first 14 residues ofthe SarR protein in the amino-terminus were determined bymicrosequencing at the Core Facility at our institution. A blast searchof the S. aureus genome data bank at TIGR revealed a partial ORF of 47amino acids. Using these data, a 141-bp fragment with two degenerateoligos was amplified by PCR.

(5′-ATG T/A C/G A/T AAAAT T/C AA T/C GATAT T/C AA T/C GATTTT-3′) (SEQ.ID NO: 4);

(5′-ATT T/A G/C A/T T/C TC T/A G/C A/T A/T C GIT T/C AA A/G AT A/G TGA/G TT T/C AA-3′) (SEQ. ID NO. 5) The PCR fragment was cloned into thevector pCR2.1 (Invitrogen). Southern hybridization of enzyme-restrictedchromosomal DNA of the parental strain RN6390 with a radiolabeled 141-bpDNA probe revealed a single ˜4 kb Cla I-digested hybridizing fragment.To clone this fragment, ClaI-digested chromosomal DNA in the range of3-5 kb was resolved in an agarose gel, excised, purified and ligated tothe ClaI site of pACYC177 in E. coli DH5α.

Positive-reacting clones were identified, all containing the ˜4 kb ClaIfragment. One of these clones was sequenced, revealing a 345-bp ORF withidentity to the partial 47 amino acid sequence of sarR as predicted fromthe S. aureus genome.

Deletion and insertion mutagenesis was performed with the StratageneQuick Change kit to introduce a deletion and a mutation concomitantlyinto the sarR gene. In brief, the ˜4 kb Clal DNA fragment containing thesarR gene in recombinant pACYC 177 was cloned into pBluescript to serveas a template for mutagenesis. The following oligonucleotide [SEQ ID.NOs: 6 and 7](5′-²²GCATGAAAAAGATATCGGGCATTT⁴⁵⁻³³⁸GTGAGTCTAACGATATCTCATCTAAA³⁶³ -3′)                       T   T                          Aand its complement were used to construct a deletion and to introduce anexogenous EcoRV restriction site into the sarR gene (restriction siteunderlined, with the native nucleotides displayed below, intact sarRgene from nt 208-555). After amplification with the recombinantpBluescript template, the PCR product was digested with DpnI to removemethylated template DNA (i.e. pBluescript with the native sarR gene) andtransformed into XL1-Blue cells to select for ampicillin-resistantcolonies. Successful deletion and mutation in the resultant clones wereconfirmed by restriction analysis with EcoRV and finally verified byautomated DNA sequencing. The ermC gene was then ligated to the EcoRVsite of the mutated construct. The fragment containing an ermCreplacement of the sarR gene was cloned into the temperature sensitiveshuttle vector pCL52.1 (57) which was then transformed into RN4220 byelectroporation (58) followed by transduction into RN6390 with phage Φ11as described (15). Transductants were selected at 30° C. on erythromycinand tetracycline-containing plates.

S. aureus RN6390 harboring the recombinant pCL52.1 was grown overnightat 30° C. in liquid medium in the presence of erythromycin, diluted1:1000 in fresh media and propagated at 42° C., a non-permissivetemperature for the replication of pCL52.1. This cycle was repeated fourtimes and the cells replicate-plated onto 03GL plates containingerythromycin and erythromycin/tetracycline to select fortetracycline-sensitive but erythromycin-resistant colonies, representingmutants with double-crossovers. The mutations were confirmed by Southernhybridization with sarR and ermC probes.

Southern Blot Hybridization. Chromosomal DNA of assorted staphylococcalspecies was isolated from lysostaphin-treated cells as previouslydescribed (15), restriction-digested, resolved in agarose gels andtransferred onto a Hybond N+ membrane (Amersham, Arlington Heights,Ill.). Hybridization was performed under high stringency conditions with³²P-labeled DNA probes as described (15). The blots were subsequentlywashed and autoradiographed.

Purification of proteins. The intact 345 bp sarR gene was amplified byPCR using RN6390 chromosomal DNA as the template and primers containingflanking restriction sites (NdeI and BamHI) to facilitate cloning intoexpression vector pET11b (Novagen). The recombinant plasmid containingthe sarR gene was transformed to E. coli BL21(DE3)pLysS. Enhancedexpression of SarR was induced by adding IPTG(isopropyl-1-thio-b-D-galactopyranoside) to a 2 L growing culture (37°C.) at an OD₆₅₀ of 0.7. After 4 hrs of additional growth, cells wereharvested, resuspended in buffer (25 mM Tris-Cl, 1 mM EDTA, pH 8.0, 100mM NaCl, 10% sucrose and 1 mM DTT), flash-frozen and thawed twice andclarified by centrifugation at 4° C. (45,000 rpm for 1 h). Afterprecipitation with 80% ammonium sulfate, the pellets were dissolved inbuffer A (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 10% glycerol,and 1 mM DTT), dialyzed against buffer A, applied to a Resource-Q columnin an AKTA purifier (Pharmacia, Piscataway, N.J.). The flow-through wasre-applied to a Resource-S column and eluted with a NaCl gradient. Thefractions were analyzed in a 12% SDS-polyacrylamide gel. Fractionscontaining the putative SarR protein were pooled, dialyzed againstbuffer A with 40% glycerol and stored at −80° C. The authenticity of theSarR protein was confirmed by determining the N-15 residues withmicrosequencing. The concentration of the purified protein wasdetermined with the Bio-Rad Protein Assay solution (Bio-RadLaboratories, Richmond, Calif.), using BSA as the standard.

Production of anti-sarR monoclonal antibodies. Purified SarR protein wasused to immunize two BALB/c×SJL/J (F1 cross) mice (100 μg each) toobtain monoclonal antibodies as described (32). The titers of the immunesera were determined by an ELISA in which diluted sera were added tomicrotiter wells precoated with SarR (5 μg/ml) as described by Jones etal. (32). After splenic fusion, antibodies from limited dilutions werescreened by an ELISA with immobilized SarR protein. Monoclonalantibodies were then purified from culture supernatants with a proteinA-agarose column as described (32).

RNA isolation and Northern analysis. Overnight cultures of S. aureuswere diluted 1:50 in CYGP broth with appropriate antibiotics and grownto mid-log (OD₆₅₀=0.7 with an 18 mm borosilicate glass tube), late-log(OD₆₅₀=1.1) and postexponential phases (OD₆₅₀=1.7). The cells werepelleted and processed with 1 ml of Trizol (Gibco-BRL, Gaithersburg,Md.) in combination with 0.1 mm diameter sirconia-silica beads in a FastPrep reciprocating shaker (BIO101, San Diego, Calif.) as described (12).Ten micrograms of total cellular RNA from each sample waselectrophoresed through a 1.5% agarose-0.66M formaldehyde gel in MOPSrunning buffer (20 mM MOPS, 10 mM sodium acetate, 2 mM EDTA, pH 7.2).RNA was transferred onto Hybond N⁺ membranes (Amersham) under mildalkaline conditions by using a Turboblotter system (Schleicher andSchuell, Keene, N.H.) as described by the manufacturer. RNA was fixed tothe membrane by baking at 80° C. for 1 hr. For detection of specifictranscripts, gel purified DNA probes were radiolabeled with ³²P-dCTP byusing the random-primed method (Ready-To-Go labeling kit, Pharmacia) andhybridized under aqueous conditions at 65° C. The blots weresubsequently washed and autoradiographed.

Promoter fusion analysis with the gfp_(uvr) reporter gene. To confirmthe effect of the sarR mutation on sarA promoter activities, sarApromoter fragments (P1, P2, P3 and combined P2-P3-P1) (39) were clonedinto shuttle vector pALC1484, which is a derivative of pSK236 containingthe recombinant gfp_(uvr) gene. The gfp_(uvr) gene was constructed byintroducing a S65T mutation into gfp_(uv) (Clontech, Palo Alto, Calif.),thereby facilitating a shift in the excitation maxima from 395 to 488 nm(26). The sarA promoter fragments were then cloned into pALC 1484,upstream of the gfp_(uvr) reporter gene. After sequence confirmation,the recombinant pALC1484s were then electroporated into RN4220 andtransduced into S. aureus strains RN6390 and its isogenic sarR mutant(15).

The activities of sarA promoter fragments linked to the gfp_(uvr)reporter gene in RN6390 and its isogenic sarR mutant were assayed byflow cytometry. Bacterial cell suspensions obtained at different partsof the growth cycle were analyzed in a FACScan (Becton Dickinson,Franklin Lakes, N.J.). After filtering bacterial samples through a 5 mμfilter to remove large aggregates, bacteria were detected by sidescatter as described by Russo-Marie et al. (56). Fluorescence and sidescatter data were collected with logarithmic amplifiers. Thefluorescence data were reported in fluorescence units as specified bythe instrument (FACScan).

To obtain more quantitative fluorescence data, each of the abovegfp_(uvr) reporter constructs was diluted 1:100 from overnight culturesinto fresh CYGP medium and, beginning at the second hour, sampled hourly(200 μl) for 10 h to encompass the growth cycle from log to stationaryphases. The samples were analyzed for total fluorescence and opticaldensities (605 nm) in microtiter wells in a multi-purpose fluorescencespectrophotometer (FL600, BioTek Instrument, Winooski, Vt.). Thefluorescence units and optical densities were given as reported by theinstrument. The background was ˜200-300 fluorescence units, withvariations of less than 100 units between duplicate samples.

Cell extracts preparation and Western analysis. Cell-free extracts frommidlog, latelog and early stationary phases (representing OD₆₅₀ of 0.7,1.1 and 1.7, respectively, in an 18 mm borosilicate tube) were preparedfrom RN6390, the isogenic sarR mutant and other staphylococcal strains.Cells were grown in CYGP broth (50 ml) supplemented with the appropriateantibiotics. After pelleting, the cells were resuspended in 0.5 ml ofTEG buffer (25 mM Tris-HCl, 5 mM EGTA; pH 8.0) and cell extracts wereprepared from lysostaphin-treated cells as described by Mahmood andKhan-(37).

Equivalent amounts of cellular proteins were separated in 12% sodiumdodecyl sulfate (SDS) polyacrylamide gels and transferred ontonitrocellulose membranes as described (61). The blots were incubated atRT with 1:1000 or 1:2000 dilution of anti-SarR or anti-SarA monoclonalantibodies for 3 h followed by another hour of incubation with a1:10,000 dilution of goat anti-mouse alkaline phosphatase conjugate(Jackson ImmunoResearch, West Grove, Pa.). Immunoreactive bands weredetected as described by Blake et al. (3). SeaBlue prestained proteinstandards (Novex, San Diego, Calif.) were used for molecular weightestimations.

Gel shift analysis and DNaseI footprinting. Gel shift assays wereperformed to determine the interaction of purified SarR with sarApromoter fragments. DNA fragments were end-labeled with -³²P ATP byusing polynucleotide kinase. Labeled DNA fragments were incubated at RTfor 20 min with the indicated amounts of purified protein in 25 μl ofbinding buffer (25 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 75 mM NaCl, 1 mMDTT, 5% glycerol) containing 0.5 μg of calf thymus DNA. The reactionmixtures were analyzed by nondenaturing polyacrylamide gelelectrophoresis as described (19). The band shifts were detected byexposing dried gels to film.

Footprinting assays with linear DNA template and DNase I were performedusing a modification of the method previously described (21). A 49-bpfragment upstream of the sarA P2 promoter region (39) was cloned intothe BamHI site of pUC18, yielding pALC926. A 109-bp EcoRI/HindIIIfragment from pALC926 was gel-purified and end-labeled with ³²P. PCRfragments containing sarA P1 (nt 531-859 and nt 620-859) and P3 (nt364-525) promoter regions were also used in footprinting reactions. Tolabel these PCR products, only one of the primers was end-labeled with-³²P in the amplification reactions, yielding PCR products labeled atone end. For the assay, the binding reactions were carried cut in a 100μl reaction volume containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mMMgCl2, 1 mM CaCl2, 2 mM DTT, 10 μg of BSA, 0.4 μg of calf thymus DNA,template DNA and varying amounts of the SarR protein at RT for 30 min.DNasel (0.02 unit) (Boehringer Mannheim, Indianapolis, Ind.) was addedand allowed to incubate for 1 min at RT. The reaction mixtures were thenextracted with phenol/chloroform. DNA was ethanol-precipitated,resuspended in loading buffer (98% formamide, 10 mM EDTA pH 8.0, 0.025%(w/v) xylene cyanol FF and 0.025% (w/v) bromophenol blue) and analyzedon a 6% denaturing polyacrylamide sequencing gel. The positions of theprotected regions were identified by comparing the footprint with theA+G sequencing ladder of the same fragment (38).

Results: The sarA promoters are differentially expressed during thegrowth cycle, with P1 and P2 promoters being most active during theexponential phase and the P3 promoter activated postexponentially (39).Because of the complexity in promoter activation and the ensuingexpression of sarA, the promoter region upstream of the sarA gene mayserve as a binding site for one or more trans acting factors (2,39).Taking advantage of a P2 promoter sequence (2) that shares homology witha region upstream of the sar P1 promoter (FIG. 1A), a DNA-specificcolumn, containing the 49-bp P2 promoter sequence, was used to partiallypurify a ˜12 kDa protein with binding properties to sarA promoterfragments (39). In the schematic shown in FIG. 1A, the positions of thetranscription starts (−146, −409 and −711 bp upstream of the translationstart) for P1, P3 and P2 promoters are depicted according to publishedsequence (2). The P1, P3 and P2 transcripts have previously beendesignated as sarA P1, sarA P3 and sarA P2 transcripts. The 49-bpsequence outlined was used to construct a DNA-specific column asdescribed (27). The relative positions of the sarA promoter fragmentsused in gel shift and footprinting studies are indicated (filled boxes)while the promoter fragments for the GFP transcriptional fusion assaysare marked as empty boxes.

To further characterize this protein and to investigate its regulatoryfunction, the sarR gene product was cloned and characterized usingbiochemical, immunological and genetic approaches.

Cloning and sequence analysis of the sarR gene. To clone the geneencoding SarR, we blotted the ˜12 kDa protein onto a PVDF membrane forN-terminal sequencing. The first 14 amino acids wereX(K)IND(I)NDLVNA(S/T)F, (Seq. ID NO.: 8) with X being an unknown residuewhile those residues in parenthesis carried a putative assignment. Insearching the databank of the partially released S. aureus genome(www.tiger.org), we obtained a partial ORF of 47 amino acid sequencethat corresponds to the N-terminal sequence of the 12 kDa protein. Byusing two degenerate oligonucleotides of 30-nt each, a 141-bp fragmentwas amplified to probe a chromosomal digest of S. aureus strain RN6390,thus allowing identification of a ˜4 kb ClaI hybridizing fragment. Aplasmid DNA library containing ˜3-5 kb ClaI fragments constructed inpACYC177 (26) was then screened with the 141-bp PCR-generated probe. Apositive clone (pALC1361) yielding a ˜4-kb insert at the ClaI site ofpACYC177 vector was identified. In determining the sequence of theinsert, and comparing the insert sequence with that of the 141-bp probe,the DNA sequence of the putative gene sarR was obtained (FIG. 1B)(GenBank accession #AF207701). The predicted SarR protein contains 115amino acids, with a predominance of charged residues (34%) and apredicted molecular size of 13,689 daltons. The sarR gene has a putativeShine Dalgarno sequence (AGGAGTGG) (SEQ. ID NO: 9) lying 7-bp upstreamof the translation start, with typical initiation (ATG) and terminationcodons (TAA). To ascertain the transcription start site and the putativepromoter boxes, the 5′-end of the sarR transcript was mapped by primerextension, using an internal primer of the non-coding strand positionednear the N-terminus of the sarR coding region. The transcriptioninitiation site is located 88-bp upstream of the translation start,thereby allowing identification of the putative −10 and −35 promoterboxes as TAGAAT (SEQ ID. No. 10) and TTACCG (SEQ ID. No. 11),respectively (FIG. 1B).

In searching the GenBank for related proteins, the entire SarR proteinshares sequence similarity with SarA (SEQ. ID NO. 3), with a highprobability score of 1.8e-⁷ (FIG. 1C). There were also otherSarR-homologs in the S. aureus database (University of Oklahoma S.aureus genome database). Like SarA, the SarR protein has a deduced basicpI (9.23). The sequence similarity between SarR and SarA is 51%, with28% identity (FIG. 1C). In limiting the homology to specific regions,residues 52-75 of SarR were found to share homology with residues 54-77of SarA. which, in turn, has a limited but regional sequence similarityto the DNA binding domain of VirF (residues 175-198), a transcriptionregulator of virulence gene expression in Shigella flexneri (18, 25).

Over-expression of SarR and production of monoclonal antibodies: Toobtain a large amount of SarR, the sarR gene was cloned into pET11b andthe gene product was over-expressed under an IPTG-inducible promoter inE. coli BL21. The expression, purification, and the purity of the SarRprotein are shown in FIG. 2. Equivalent volumes of protein fractionsduring the purification process was applied to a 12% SDS-polyacrylamidegel. FIG. 2, Lane 1, whole cell lysate of E. coli containing pALC1357(pET11b with the sarR gene); FIG. 2, lane 2, supernatant of the celllysate after clarification by centrifugation; FIG. 2, lane 3,supernatant before 40% ammonium sulfate precipitation; FIG. 2, lane 4,pellet resulting from 40% ammonium sulfate precipitation; FIG. 2, lane5, pellet from 80% ammonium sulfate precipitation; FIG. 2, lane 6, fallthrough of the redissolved 80% ammonium sulfate precipitant as appliedto a MonoQ column (Pharmacia); FIG. 2, lane 7, fall through from theMonoS column (Pharmacia); FIG. 2, lane 8, NaCl elution from the MonoScolumn. N-terminal sequencing confirmed the identity of the purifiedSarR protein. The SarR protein was expressed primarily in the cytosolicfraction (FIG. 2, lane 2). After 80% ammonium sulfate precipitation(FIG. 2, lane 5), the redissolved proteins were dialyzed and applied toan anion exchange column (Resource-Q, Pharmacia), only to be found inthe fall-through (FIG. 2, lane 6). The flow-though was then applied to acation exchange column (Resource-S column, Pharmacia) and eluted with asalt gradient. Using this purification scheme, SarR was purified to nearhomogeneity (FIG. 2, lane 8). The authenticity of SarR was confirmed byN-terminal sequencing. The purified SarR was then used to immunize micefor the production of anti-SarR monoclonal antibodies. Three monoclonalantibodies, designated 2A7, 2C7, and 5E4 were obtained. Despite thesimilarity between SarR and SarA, cross-reactive studies indicated thatanti-SarR monoclonal antibodies only reacted with SarR and not SarA onimmunoblots.

The Existence of sarR in Other Staphylococcal Strains and OtherProkaryotes

PCR analysis was conducted to determine if the SarR gene is present inother S. aureus strains, other staphylococcal species and in otherbacteria. This analysis demonstrates that the sarR gene is present inall strains of S. aureus as well as in S. saprophyticus and S.haemolyticus. Blast searches confirm this result.

Binding of SarR to sarA Promoter Fragments by Gel Shift and FootprintingAssays.

The interaction between SarR and various sarA promoter fragments wasexamined with gel shift and footprinting assays. Accordingly, purifiedrecombinant SarR from E. coli was used in gel shift assays with assortedDNA fragments of the sarA promoter region including P2 (nt 1-196, P3 (nt364-525), and P1 (nt 531-859). The mobility of the labeled DNA fragmentsbecame more hindered with increasing concentrations of SarR in gel shiftassays (FIG. 4). In the assays, Increasing amounts (30, 60, 100, 150,200, 250, 300 ng) of purified SarR were applied to the reactionmixtures. In competition assays, 50 and 100 fold excess of unlabeled DNAfragments were added. The unusual laddering pattern of the band shiftswas observed with all three sarA promoter fragments. One plausibleexplanation is that each of the sarA promoter fragments may containmultiple binding sites. Alternatively, the binding of SarR in multimericform to a common site or multiple sites within each of the sar promoterfragment is plausible. An analysis of the relative binding of SarR andSarA to the sarA PI promoter indicates that the amount of SarA requiredto completely retard the mobility of 2-5 ng of radiolabeled sarA P1fragment is 10 times more than that of SarR, thus demonstrating thehigher avidity of SarR than SarA for the sarA P1 promoter fragment.

To determine the binding site of SarR and to verify the specificity ofbinding to the sarA promoter region, DNase I footprinting analysis wasperformed. To elucidate the SarR binding site, a 109-bp EcoRI-HindIIIfragment derived from pUC18 containing the 49-bp sequence (39) wasend-labeled at the EcoRI or HindIII sites separately and subjected toDNaseI footprinting with assorted concentrations of SarR. The sequencewas deduced from G/A ladder reactions run in parallel following thestandard method (26). The following amounts of SarR were applied to thesarA P2 and P1 reactions: 30, 60 and 100 ng, With sar P1′, only lanescontaining 30 and 60 ng of SarR protein were shown. The binding sites ofSarR on the sarA P3 promoter was also mapped: 373TTACTAAATTAAAAAAATTA⁴⁰² (SEA. ID NO. 12) (2). Analysis of the footprintof the plus strand (EcoRI site end-labeled) (FIG. 5A) disclosed theprotected region (nt⁸¹ TAAATTAA TGTTATTTTTTAATAATTAA ¹⁰⁸) (SEQ ID.NO.13) (2) to be extremely A/T rich (96%), thus implying specificbinding of SarR to this region but not to the more GC-rich polylinkerregion of pUC18, even when higher concentrations of SarR were used inthe assay. A similar protection site was also found for the minus strand(HindIII site end-labeled). In analyzing the SarR protected region, thesite was found to consist of a 7-8 sequence (TAAATTAA (SEQ ID. NO. 14),With the last base variable) conserved in both strands (e.g.¹⁰¹ATAATTTA¹⁰⁸ (SEQ ID. NO. 15) being complement of TAAATTAA) andthroughout the sarA promoter region (39).

The binding of SarR to other sarA promoter regions was also determined.It has been shown that an inverted repeat region (nt 553-593) upstreamof the sar P1 promoter may play a role in repressing sarA P1transcription (39). Recognizing that SarR binds to a large P1 fragmentin gel shift assays (FIG. 4), a footprinting analysis was performed withtwo different DNA fragments upstream of the sarA P1 promoter [329-bp (nt531-859) and 240-bp (nt 620-859)(2)]. Using ³²P end-labeled sensestrand, the SarR-protected region on the 329-bp sarA P1 promoterfragment was found to comprise several regions including nt 551 to 553,556 to 575, 582 to 603 (⁵⁸⁶TAAATTAT ⁵⁹³) (SEQ ID. NO. 16), 620 to 640(FIG. 5B). In analyzing the smaller 240-bp P1 fragment, four additionalprotected regions, downstream of the above binding sites, wereuncovered: nt 633 to 640, 643 to 667, 673 to 678, and 687 to 708 (FIG.5C). Thus, the inverted repeat region (nt 553-593), which has previouslybeen shown to play a putative role in repressing P1 transcription (39),is also part of the SarR binding sites. The SarR binding site on thesarA P3 promoter was also uncovered:

-   -   ³⁷³ ¹⁷TTACTAAATTAAAAAAATTA⁴⁰² (SEQ I.D. NO. 17) (2). In        comparing the broad binding sites protected by SarR, a common        feature is their highly AT-rich nature. More remarkably, the 7-8        bp conserved sequence (TAAATTAA) (SEQ. ID NO:14) was included        within the SarR binding sites in each of the sarA promoter        fragments (P2, P1 and P3).        Expression of the sarR Gene in RN6390 and its Isogenic SarA and        agr Mutants:

During the growth cycle, the major sarA gene product such as SarApartially mediates its effect by binding to the agr promoter toinfluence RNAII and RNAIII transcription. To ascertain if the sarR geneis modulated by sarA or agr (i.e. acting downstream of the sarA or agrregulatory cascade), sarR transcription in parental strain RN6390 andits isogenic agr and sarA mutants was assayed. To ensure that comparableamounts of total cellular RNA were applied to each lane, ribosomal RNAbands stained with ethidium bromide were compared among the lanes (FIG.6A). Ten fig of total cellular RNA was applied to each lane. The sarRprobe was a 345-bp fragment (nt 208-552). The OD_(650nm) of 0.7, 1.1 and1.7 represent mid log, late log and early stationary phase,respectively, as predicted from the growth cycle. As displayed in FIG.6B, the transcription of sarR in RN6390 could be detected in mid logphase and was maximally expressed during the postexponential phase.Accounting for minor experimental variations, the observation that sarRtranscription was not significantly altered in sarA and agr mutantsindicated that sarA and agr did not regulate sarR as one would expect ifthese regulating loci lie downstream of sarR. This is consistent withthe finding (described below) that a mutation in sarR affects sarA andagr transcriptions.

The expression of the SarR protein during the growth cycle byimmunoblots was also determined. Using anti-SarR monoclonal antibody 2C7(1:1000 dilution), an immunoblot of cell-free extracts of RN6390 derivedfrom cells grown to late-log (OD₆₅₀=1.1) and postexponential phases(OD₆₅₀=1.7) was probed. Employing ˜25 μg of cellular proteins in eachlane, it was found that the expression of SarR corresponds quite wellwith the pattern of sarR transcription, with SarR detectable at late-logphase, and maximal during the postexponential phase (FIG. 6C). In FIG.6C, Each lane contains cell-free extracts (25 μg each) of RN6390 cellsgrown to late-log and early stationary phases. Cells at mid-log phaseexpressed little SarR; as expected, SarR was not detected in the sarRmutant ALC1713 (data not shown). The positive control lane contains 0.1μg of purified SarR protein.

The expression of sarA in a sarR mutant: To demonstrate that the SarRprotein likely modulates sarA expression by virtue of its binding to thesarA promoter region, a sarR deletion mutant was constructed byreplacing the sarR gene with an ermC gene in strain RN6390. Northernanalysis confirmed that the transcription of sarR was disrupted in sarRmutant ALC1713. To analyze the effect of sarR on individual sarApromoters, P2 (nt 1-180 plus 197-bp upstream), P3 (nt 364-525), P1 (nt620-859) and the combined (or native) P2-P3-P1 promoters (nt 1-859 plus197-bp upstream) (2, 39) upstream of the gfp_(uvr) reporter gene werecloned in shuttle plasmid pALC 1484.

Flow cytometry was used to evaluate promoter activity, demonstrated thatthe sarA P1 and the combined P2-P3-P1 promoters were more active in thesarR mutant than the parental control. Mean fluorescence was 5.01±0.29(log scale) in RN6390 vs 5.84±0.13 in sarR mutant and 5.49±0.21 inRN6390 vs 8.44±0.24 in the mutant, for P1 and combined promoters,respectively, during the postexponential phase. However, the relativeweakness of the sarA P3 and P2 promoters as compared with the P1promoter (˜20 to 30 fold less than of P1) (39), coupled with therelative stability of the GFP reporter, rendered flow cytometry lessuseful to record small variations in GFP expression during the growthcycle among 10,000 organisms gated for this experiment. Notsurprisingly, we failed to detect differences in activation of theweaker sarA P3 and P2 promoters between the parent and the isogenic sarRmutant by flow cytometry (39). More specifically, the level of P2 and P3activation as detected by flow cytometry was only slightly abovebackgrounds.

To obtain more quantitative fluorescence data for a larger number ofbacterial cells, a multi-function fluorescence spectrophotometer in amicrotiter format was used (FL600 from BioTek Instrument, Winooski, Vt.)to measure optical densities and total fluorescence of samples (200 μl)obtained serially during the growth cycle. To minimize the variation influorescence attributable to cell density, fluorescence per OD againstoptical densities was plotted over a 10 h period (extending from log tostationary phase). The data showed that the sarA P1-GFP fusion activityin the sarR mutant was higher than the parental strain (RN6390)throughout the growth cycle (FIG. 7A). FIG. 7A illustrates recombinantshuttle plasmid pALC1484 containing the sarA P1 promoter linked togfp_(uvr) (excitation maxima 488 nm) in strain RN6390 (μ) and itsisogenic sarR mutant ALC1713. A negative control (RN6390 containing onlypALC1484 without any promoter fragment) did not display any significantbackground fluorescence (˜300 fluorescence units as the background, datanot shown). Cells were obtained hourly (200 μl each in duplicate) duringthe growth cycle (from the 2^(nd) to the 10^(th) h after an initialdilution of 1:100 in fresh medium) to obtain fluorescence and opticaldensities (OD) in the same instrument. The data were presented as theaverage of reported fluorescence per OD in triplicate samples plottedagainst the mean of the optical densities. The error bar was too smallto be discerned, typically less than 100 fluorescence units. Thisexperiment has been repeated at least thrice. One representativeexperiment was shown. The early decline in fluorescence for both strainswas likely to be attributed to the carryover effect of the GFP from theovernight inoculum. Following the decline, the highest level of GFPfusion in the mutant occurred late in the stationary phase (at ˜7-10 hafter initial culture dilution) (FIG. 7A), at a time when the sarRtranscript level and the expression of SarR in the parent were highest(FIGS. 6B and 6C). The level of GFP fusion for the sarA P1 promoter inthe parental strain declined after the initial dilution, but higher thanthe background (background fluorescence ˜300 units), during the growthcycle. This finding is attributable to a steady decrease in P1 promoteractivation (per bacterial cell) in the parental strain as the cell cycleprogressed. Additionally, a lack of contribution from upstream promoters(i.e. P3 and P2) to modulate P1 activity in this promoter fragment mayconceivably play a role. Similar studies were also conducted for thecombined or native sarA P2-P3-P1 promoter linked to the gfp_(uvr)reporter. In this instance, the combined promoter activity in the sarRmutant was also higher than that of the parent. FIG. 7B illustrates aplot similar to FIG. 7A except that the combined sarA P2-P3-P1 promoterfragment was used in place of the P1 promoter in the recombinantpALC1484 containing the gfp_(uvr) reporter gene. Similar assays werealso performed with the individual sar P2 and P3 promoters linked to thegfp_(uvr) reporter in the isogenic pair, but failed to detect anydifferences in GFP_(uvr) expression between the parental strain and thesarR mutant. However, the level of fluorescence associated withindividual P2 and P3 promoters was very low and only slightly abovebackgrounds. As with the P1 promoter, the level of activity for thecombined promoter decreased after initial culture dilution for bothstrains and then increased during the postexponential phase. Ofinterest, the increase in combined promoter activity with growth in theparental strain suggests that the sequence element upstream of P1 mayhave contributed to the overall increase in combined promoter activityduring the postexponential phase. However, no differences influorescence for the P2 or the P3 promoter GFP fusions between the sarRmutant and its isogenic parent were detected. Notably, the fluorescenceof the P2 and P3 promoters was only slightly above background. Thus,this shows that the fluorescent assays may not be sensitive enough todetect subtle differences in P2 and P3 promoter activities. Todemonstrate that an intact sarR gene has a negative impact on theexpression of SarA, the major sar regulatory molecule, cell-freeextracts of the isogenic sarR strains during various stages of thegrowth cycle were obtained. Using cell extracts (25 μg of proteins each)of the sarR mutant obtained at different phases of the growth cycle, animmunoblot with anti-SarA monoclonal antibody 1D1 (20) was probed. Asshown in FIG. 8A, the sarR mutant expressed higher levels of SarAprotein than the isogenic parent at optical densities representingmid-log, late-log and stationary phases. FIG. 8A shows an immunoblot ofthe cell extracts (5 μg of protein each) of RN6390 and the sarR mutant(harvested at midlog, late-log and stationary phase) probed withanti-SarA monoclonal antibody 1D1 at 1:2000 dilution. The positivecontrol lane contains 0.5 μg of purified SarA. Similar results wereobtained with 25 μg of protein per lane. Notably, in both the parentaland the mutant strains, SarA expression was maximal during the late-logphase and tapered toward the stationary phase. This shows that thereduction in SarA expression in the parental strain during thestationary phase is explained in part by increased proteolytic activityand hence processing of SarA in stationary cells (39, 51). Additionalimmunoblots with increased amounts of cell extracts (at 25 μg each) frommid-log, late-log and stationary phases also confirmed higher expressionof sarA in the sarR mutant as compared to the parental strain. Takentogether, these data demonstrate that SarR is a DNA binding protein thatbinds to the sarA promoter to down-regulate sarA expression.

The level of SarA has been shown to correlate with the extent of agractivation (20). Northern analysis of sarR mutant ALC 1713 with an agrA(RNAII) probe confirmed a higher level of RNAII expression as comparedwith the parental control (FIG. 8B). Collectively, these datademonstrate that SarR down-regulates sarA expression, by binding to thesarA promoter to down-modulate sarA P1 transcription. The end result isthe modulation of target genes (e.g. agr) downstream of the sarAregulatory cascade.

Protein expression, purification and crystallization: The intact 345-bpsarR gene was amplified by PCR using chromosomal DNA from S. aureusstrain RN6390 as the template and primers containing flankingrestriction sites (NdeI and BamHI) to facilitate cloning into anexpression vector pMAL-c2 (BioLabs, Beverly, Mass.) modified bytruncating 21 residues from the linker region that connects SarR andMBP. The recombinant plasmid containing the sarR gene was transformed toE. coli BL21(DE3)pLysS. Enhanced expression of sarR-MBP fusion wasinduced by adding IPTG (isopropyl-1-thio-b-D-galactopyranoside) to a 4 Lgrowing culture (37° C.) at an OD₆₅₀ of 0.7. After 4 hrs of additionalgrowth, cells were harvested, resuspended in buffer (50 mM Tris-HCl, 1mM EDTA, pH 7.4, 300 mM NaCl, 5% glycerol and 1 mM DTT) and subjected tocell lysis through a continuous-flow French press. After a 20,000×gspin, the soluble fraction was loaded onto an amylose resin affinitycolumn (10 ml) and the SarR-MBP fusion protein was eluted with 10 mMmaltose. The protein was loaded onto a MonoQ (Pharmacia) ion-exchangecolumn. After elution with a NaCl gradient (0.1 to 0.5 M) the fractioncontaining the protein was found to be homogeneous as determined by aCoomassie stained SDS-polyacrylamide gel. The concentration of thepurified protein was determined with the Bio-Rad Protein Assay solution(Bio-Rad Laboratories, Richmond, Calif.), using BSA as the standard. TheSarR-MBP fusion protein (15 mg/ml) was crystallized by vapor diffusionagainst a solution of 5 mM β-mercaptoethanol, 100 mM Na acetate, 100 mMNaCl, pH 4.6, and 18-22% PEG monomethyl ether 2000. Forcryo-crystallography, crystals were soaked in steps of increasingglycerol concentration (5% each step every 30 min) and finally into 20%glycerol before flash-freezing.

Structure Determination and Refinement: Data were collected on a RigakuR-axis IV system and beamline 5.0.2 at ALS. Data processing wasperformed with DENZO and SCALEPACK (50). The initial phases wereobtained by a molecular replacement solution using the available MBPstructure (53) and program AMoRe (45). 2Fo-Fc and Fo-Fc electron densitymaps were calculated by CNS (6). Map interpretation and model buildingwere done using the program O (33). The map was improved by cycles ofrefinement using CNS with NCS constraints. A final refinement wasperformed with relaxed NCS-restraints as shown in Table 2 below. Thefinal model contains residues 1-115 for molecule one of SarR, residues1-115 for molecule two of sarR (FIG. 9B), residues 1-372 for both MBPmolecules (FIG. 9A), two maltose molecules, and 190 water molecules.Stereochemical values are all within or better than the expected rangesfor a 2.3 Å structure, as determined using PROCHECK (35). TABLE 2Summary of Crystallographic Analysis Diffraction Data and MR statisticsResolution R_(merge) ^(b) Reflections Completeness Crystal^(a) (Å) (%)Measured/Unique (%) Solutions^(e) C.O.^(e) R^(e) Native I 2.8 4.362396/20967 78.0 2 47.2 46.6 Native II 2.3 2.7 77545/37403 96.5Refinement (Native II) Resolution (Å) 20-4.58 3.65 3.19 2.90 2.69 2.532.40 2.30 Total No. reflections 5696 5656 5512 5198 4788 4180 3454 270237186 R-factor^(c) 15.27 20.16 29.30 33.14 36.95 38.04 38.86 41.56 23.24Free R-factor^(d) 21.94 24.45 32.45 38.65 39.36 38.79 35.91 38.02 28.22r.m.s. Deviations; Bonds, 0.009 Å; Angles, 1.4° ^(a)Crystal spacegroupP1; a = 64.7 Å, b = 70.6 Å, c = 75.5 Å, α = 65.7°, β = 67.2°, γ = 69.6°,two sarR-MBP monomers/unit cell${\quad^{b}R\text{-}{merge}} =  {{{100 \times {\sum\limits_{j}\quad I_{j}}} -} < I >} \middle| {/{\sum\limits_{j}\quad{I_{j}\quad{with}\quad{Bijovet}\quad{pairs}\quad{treated}\quad{as}\quad{equivalent}}}} $^(c)R-factor = Σ/Fobs · F_(calc)|/ΣF_(obs) for all amplitudes withF/σ(F) ≧ 2 measured in the indicated resolution bin ^(d)Free R-factorwas calculated with 5% of the data in each bin. ^(e)Molecularreplacement solutions, C.O. was correlation coefficients, and R, aredefined in Amore (18)

Overall Structure: The structure of SarR-MBP fusion protein shows thatthe SarR dimer is located at the top of the two individual MBPmolecules, connected by two flexible loop regions between SarR and MBP.The positions of the MBP molecules suggest that they did not influencethe structure of SarR dimer (FIG. 9A). The overall structure of the SarRmonomer consists of five a helices, three short β strands, and severalloops (FIG. 9B). The al helix extends out from the remaining molecule,forming a ‘L’ shaped like structure with a stretch formed from theN-terminal residues of the protein. The α2 helix follows a 7-residueloop from α1, and is almost perpendicular to α1 (85°). The three βstrands, β1, β2, and β3, form an anti-parallel bundle, which is slightlytwisted. α5 follows immediately after β3. Between β1 and β2 is a longflexible region (residues 51-79), which has poor electron density in theinitial 2Fo-Fc and Fo-Fc maps, containing two helices, α3 (residue51-56) and α4 (residue 63-75) respectively, and a short turn (residue56-58). These three elements build up a typical helix-turn-helixstructural module existing in DNA-binding proteins. Homology alignmentof the SarR structure with all available structures shows that the SarRmonomer is homologous to winged helix proteins (23), such astranscription regulatory protein mota fragment (PDB code 1bja) with Zscore of 8.0, transcriptional repressor smtb activation domain (PDB code1 smt) with Z score of 7.2. Compared to winged helix proteins, “W2” isreplaced by a helix (α5) and the “W1” extends much further in the SarRmonomer (FIG. 9B). The above data shows that SarR and its family ofproteins are new members of the classic winged helix protein family.

Two L-like structures of α1 and the stretch of the N-termini cometogether forming a dimer that has an elongated, slightly bent structurewith overall dimensions of 71 Å×37 Å×34 Å (FIGS. 10A and 10B). Themonomers are related to each other by a non-crystallographic localtwo-fold axis. On the concave side and middle of the SarR dimer, thereis a canyon like structure with length of ˜35 Å, a width of ˜25 Å, and adepth of ˜10 Å (FIGS. 10A and 10B). The canyon is formed by a part ofα1, the loop that connects α1 and α2 and a part of α2 as its bed, andwith α4 from both monomers acting as the two banks (FIGS. 10A and 10B).On the convex side, all four termini (C and N-termini of both molecules)form a flat platform with the N-terminus of one monomer adjacent to theC-terminus of the other. This feature suggests that SarS, a 250 residueprotein homologous to SarA (23), functions as a heterodimer-like monomersince it contains two sections, highly similar to the SarR module, each125 residues long (17). The entire dimer can be described as threeindividual subdomain structures (FIG. 10A). Three beta strands plus α3and α4 from each molecule form subdomain 1 and subdomain 2,respectively. Those two subdomains were poorly defined in the initialelectron density map, which also was reflected by their high temperaturefactors compared to the average (89 via 69 ) in the final model, and canbe expected to have high mobility in the molecule in solution. Subdomain3, consisting of the major alpha helices α1, α2, and α5 from bothmonomers, is relatively rigid due to restrains of hydrophobicinteractions. These helices in subdomain 3 are arranged in such a mannerthat the entire subdomain looks like a twisted letter Z with therelatively flexible subdomain 1 and subdomain 2 covering its ends. TheZ-shaped scaffold, a unique structural feature for SarA family,represents a new functional protein fold (FIGS. 2A and 2B).

Dimerization Interfaces: Several lines of evidence suggest that, withthe exception of SarS, the active form of the SarA family of proteins isa homodimer. First, there is strong evidence showing that SarA exists ashomodimers in vitro as well as in vivo (60). Second, upon mixingpartially unfolded (4M urea) full length SarR protein and SarR-MBPfusion, a heterodimer product containing one copy each of SarR andSarR-MBP fusion could be separated by gel-filtration chromatography.Third, crystals of SarR-MBP fusion protein were used to solve the SarRstructure. The SarR protein exists as a dimer even in the MBP fusionform and indicating that the dimerization form is the active formexisting in vivo. Fourth, based upon the homodimer SarR structure, theinteractions between the two monomers are quite extensive, with mostresidues involved in hydrophobic interactions (FIGS. 9A and 11).Residues Leu10, Ile7, Ile 4 from one monomer and residues Phe20, Ile35,Leu 109 and Ile 113 from the other monomer form two hydrophobic cores.The dimer interface buries ˜1,500 Å² of solvent-accessible surface area(1.4 Å probe, 25) per monomer. The hydrophobic feature of SarRdimerization, indicates that the dimer of SarR in the fusion protein isalso the functional dimer in vivo. Furthermore, the L shape likestructure of α1 and the stretch of the N-terminus of SarR physicallyblock the dissociation of one monomer from its partner. This explainsthe need for extremely harsh conditions (e.g., 4M urea) in order todisrupt the dimerization of individual monomers. Fifth, deletionanalysis showed that mutated sarA, with a 15-residue truncation at itsN-terminus, exists as monomers in vitro (in solution or crystal packingforms). Finally, as shown by the sequence alignment of SarA, SarR, andSarS, most residues involved in the dimerization process are highlyconserved (FIG. 11).

DNA Binding and Bending: The DNA binding domain of SarR is conserved inthe SarA protein family. The binding domains include the helix turnhelix motif and the wing of the SarR molecule. SarA and SarR proteinscontain a high percentage of the residue Lys (39). Remarkably, most ofthe Lys residues are highly conserved between these two proteins (FIG.1). It was predicted that most of these Lys residues are involved in DNAbinding (39). Although the Lys residues are distributed throughout theentire primary sequence, in the 3D structure, most Lys residues and someArg residues are located primarily on one surface of the SarR dimer(i.e., the concave side). The electrostatic potential on this surface ofthe SarR dimer, calculated by the Grasp program (46), revealed apositively charged track on this side (FIG. 12A). Additionally, the twowinged helix motifs (domain 1 plus α2 from one monomer and domain 2 plusα2 from another monomer) are located on this side. Accordingly, thisside is apparently the site for DNA binding.

Using the program Dali to search for proteins with a structure similarto SarR, every structure with a Z score higher than 4 was manuallyexamined. It was determined that the spatial arrangement of the two SarRhelix-turn-helix modules is quite similar to those in the cataboliteactivating protein (CAP) (33, PDB code 1cgp with Z score of 4.7).Superposing the SarR winged helix motif 1 and 2 with the correspondingdomains from CAP dimer, the root mean square deviation (rmsd) of thecc-carbon backbone for motif 1 is 1.8 Å, and 2.1 Å for motif 2 (FIG.13A). Interestingly, some of the residues involved in DNA binding andbending in the CAP dimer, which interact with the major grooves, areconserved in the SarR dimer and in other members of the Sar family (FIG.1). For example, SarR charged residues that are predicted to interactwith phosphate groups on the DNA backbone, are possibly Lys 52, Lys 56,Lys 71, and Arg 82. Lys 52 and Arg 82 are absolutely conserved in SarRand in the SarA family of proteins. Charged residues predicted tocontact DNA bases are Lys 61 and Lys 67. Lys 61 is also absolutelyconserved. Therefore, this shows that the SarR dimer and other SarAfamily members have similar protein-DNA interaction as the CAP dimer.

The interaction causes the DNA to bend at two points by ˜90° (59). Thelongest direct distance for the CAP and SarR dimer surface is ˜71 Å(FIG. 12), which can hold a stretch of bent DNA with ˜27 base pairnucleotides that has length of ˜92 Å for a normal B-form (59). This isconsistent with the experimental DNA footprinting data, showing that ˜29nucleotides from the sarA promoter region were involved in binding toSarR (21). This predicted bending of the DNA when SarR binds to DNA, mayreflect a regulatory mechanism for the SarA protein family incontrolling target gene transcription.

The SarR dimer was superimposed on the CAP-DNA complex to construct amodel for a SarR-DNA complex (FIGS. 13B and C). This model suggests thatin addition to interactions of the α4 helix with the DNA major groove,SarR makes contacts with the DNA minor groove. The loop region betweenβ2 and β3 and part of the two beta strands (W1, a β-hairpin) should bequite flexible in the free SarR structure. Only slight adjustments oftheir conformations are required to position them to interactintensively with the minor groove of the DNA. Several residues thatcould be involved in the interactions are highly conserved: Asp 86, Glu87, and Arg 88 with the side chain of Arg 88 interacting with the DNAphosphate backbone and the side chains of Asp 86 and Glu 87 interactingwith bases (FIGS. 13B and C). This loop is too short in CAP and otherwinged helix proteins to have this minor groove binding function (23).One new member of one of the classes of winged helix proteins (RFX) doesmake DNA minor groove contacts, but in this case, the wing contacts themajor groove and the helix contacted the minor groove (24). Therefore,the predicted SarR type of wing-minor groove interaction appears to be aunique feature of the SarA family of proteins establishing them as athird class of the winged helix family (23,24).

The two subdomains involved in the DNA binding and bending in CAP havedifferent conformation at the absence of DNA, but are identical in thecomplex structure (23). The corresponding subdomains (1 and 2) in SarRare almost identical, but most of side chains are poorly defined. Thosetwo subdomains should have high mobility in the absence of DNA just asthese are in the CAP protein. The mobility can be reflected by theirhigh temperature factors (FIG. 9A).

Regulation Mechanism: One class of transcription-activating proteinsbears two structural motifs, namely a DNA binding and an activationdomains (52). The SarR protein was initially defined as atranscriptional repressor protein that binds the sarA promoter region(39), thus leading to reduced transcription from the sarA P1 promoter.As stated previously, the sarA P1 promoter is the predominant promoterin the sarA regulatory system. Transcriptional fusion studies indicatethat the sarA locus is auto-regulatory (21), possibly mediated by thebinding of SarA to its own promoter. The binding affinity of SarR to asarA promoter fragment is higher than its SarA counterpart (40),consistent with the idea that an activation motif might be present onthe SarA protein but not on the SarR protein, and that SarR may repressby a simple competitive displacement mechanism. A second possibility isthat SarA and SarR may form a hetero-dimer to interfere with thefunction of the SarA homo-dimer. Due to the conservation of residuesinvolved in the dimerization, this could happen in vivo. Finally, SarRmay function similarly to the bacteriophage lambda repressor (which alsohas a helix-turn-helix DNA binding motif). In this case, a slight DNAbinding site difference (one base pair shift) could turn an activator toa repressor by affecting the RNA polymerase binding (7).

The structure of the SarR protein, combined with the sequence alignmentof additional SarA family members (FIG. 11), shows that the regulatorymechanism is complicated. Residues 7, 8, and 11 appear to be importantresidues in the activation domain. When compared to the CAP, there is noisolated activation domain in the SarR dimeric structure, a calculationof the surface electronic potential revealed two negatively chargedpatches on the convex side of the SarR dimer (FIG. 12B). These patchesinclude residues Asp6 and Asp9 from molecule 1, Glu108, Glu110, andGlu111 from molecule 2. Many transcription regulators work by bindingDNA and then interacting with a component of the RNA polymerasemachinery (52). For example, CAP regulates downstream protein expressionmostly through its interactions with the C-terminal domains of alphasubunit of RNA polymerase (28). These acidic patches on the surface ofSarR may represent activation motifs that allow SarR to regulate geneexpression in a similar way. Since Glu108, Glu110, and Glu111 are notconserved among the SarA family (FIG. 11), this activity may have adifferent specificity among the family members. In this regard, we havedetermined that SarR, besides interacting with the sarA promoter, alsodirectly binds to the hla promoter (e.g. alpha hemolysin gene), thusbypassing the effect of sarA in controlling target genes.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

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1. A nucleic acid sequence which regulates the expression of virulencedeterminants in gram positive bacteria.
 2. The nucleic acid sequence ofclaim 1, wherein the bacteria is a Staphylococcus species.
 3. Thenucleic acid sequence of claim 2, wherein the bacterium isStaphylococcus aureus.
 4. The nucleic acid sequence of claim 1comprising the sequence as essentially set forth in SEQ ID NO:
 1. 5. Avector comprising the nucleic acid sequence of claim
 4. 6. A host cellcomprising the vector of claim
 5. 7. A method for identifying putativeagents which inhibit growth and infectivity of bacteria comprisingidentifying agents which enhance the expression of the nucleic acidsequence of claim 1 or the activity of a polypeptide encoded thereby. 8.The method of claim 7 wherein the bacterium is Staphylococcus species.9. The method of claim 8 wherein the bacterium is Staphylococcus aureus.10. A method of inhibiting growth and infectivity of bacteria comprisingcontacting the bacteria with an agent which enhances the expression ofthe nucleic acid sequence of claim 1 or the activity of a polypeptideencoded thereby.
 11. The method of claim 10 wherein the bacterium is aStaphylococcus species.
 12. The method of claim 10 wherein the bacteriumis Staphylococcus aureus.
 13. A pharmaceutical composition for use as ananti-bacterial agent comprising an agent which enhances the expressionof the nucleic acic sequence of claim 1 or the activity of a polypeptideencoded thereby and a pharmaceutically acceptable vehicle.
 14. Thecomposition of claim 13 which is an anti-bacterial agent against aStaphylococcus species.
 15. The composition of claim 13 wherein thebacterium is Staphylococcus aureus.
 16. An isolated polypeptide whichregulates the expression of virulence determinants in gram positivebacteria.
 17. A polypeptide encoded by the nucleic acid sequenceessentially as set forth in SEQ ID NO:
 2. 18. The isolated polypeptideof claim 16 wherein the bacterium is Staphylococcus species.
 19. Theisolated polypeptide of claim 18 wherein the bacterium is Staphylococcusaureus.
 20. A kit for identifying the presence of a sarR gene or sarRgene product comprising a means for analyzing a biological sample forthe presence of the sarR gene or the sarR gene product.
 21. A method oftreating a mammal suffering from or susceptible to a gram positivebacterial infection comprising administering a compound capable ofselective occupation of a sarA promoter receptor.
 22. A method ofscreening for lead compounds which inhibit the expression of virulencedeterminants in gram positive bacteria comprising identifying chemicalentities having structural similarities to the SarR protein sufficientto allow said chemical entities to form heterodimers with SarA protein.23. A method of screening for lead compounds which inhibit theexpression of virulence determinants in gram positive bacteriacomprising identifying chemical entities having structural similaritiesto the SarR protein sufficient to allow said chemical entities to bindsarA promoter receptors.
 24. A pharmaceutical composition comprising acompound identified by the screening method of claim 22 or
 23. 25. Apharmaceutical composition comprising-a compound identified by thescreening method of claim 22 or 23, wherein said composition is capableof inhibiting the expression of sarA.
 26. A pharmaceutical compositionfor the treatment of gram positive bacteremia comprising a SarR agonist.27. A pharmaceutical composition comprising a compound that binds to theP1 promoter region of a sarA gene.